[Non-vitamin K dependent oral anticoagulants : What is important in intensive care medicine]. / Nicht-Vitamin-K-abhängige orale Antikoagulanzien : Was ist in der Intensivmedizin zu beachten.

Since first used in 2009, non-vitamin K oral anticoagulants (NOAC) have gained world-wide acceptance. Two groups of NOAC are currently used: the direct thrombin antagonist dabigatran and three direct factor  Xa antagonists apixaban, edoxaban, and ricaroxaban. With their increasing use for prevention of thromboembolism, the probability increases that NOAC-pretreated patients are admitted to emergency departments or intensive care units.The clinical challenge in NOAC preanticoagulated patients is to adequately cope with the given anticoagulated status of such patients. Because of their short half-life, many patients will be adequately treated with a "wait and see" approach, and surgeries and interventions are postponed until anticoagulant activities have totally subsided. In the few cases where immediate action is mandated, based on appropriate risk assessments it can be decided either to take the increased hemorrhagic risk of early intervention or to transfuse factor concentrates like PPSB or FEIBA which can safely reverse the anticoagulant activities of the three factor Xa antagonists (and potentially also of dabigatran). Recently a humanized Fab antibody fragment for dabigatran, idarucizumab, has been introduced onto the market, that can immediately reverse the anticoagulant effects of dabigatran. For the reversal of dabigatran, idarucizumab is therefore the drug of choice.In addition, in some specific indications of emergency and intensive care medicine, the primary use of a NOAC can be considered advantageous. Such indications are early cardioversion in patients admitted for new episodes of atrial fibrillation and patients with acute pulmonary embolism. For the widespread use of low-molecular-weight heparins in such indications, however, the decision to use a NOAC for anticoagulant therapy is frequently postponed to the treatment phase when the stabilized patient is already treated on the general ward.

Argatroban for elective percutaneous coronary intervention: the ARG-E04 multi-center study.

UNLABELLED: The synthetic arginine-derived direct thrombin inhibitor argatroban is an attractive anticoagulant for percutaneous coronary intervention (PCI), because of its rapid onset and offset, and its hepatic elimination. Argatroban was approved for PCI in patients with heparin-induced thrombocytopenia (HIT). However, there are limited data about argatroban in non-HIT patients. The objective of this open-label, multiple-dose, controlled study was to examine the safety and efficacy of argatroban in patients undergoing elective PCI. METHODS AND RESULTS: Of 140 patients randomized to three argatroban dose groups (ARG250, ARG300, and ARG350 with 250, 300, or 350 µg/kg bolus, followed by 15, 20, or 25 µg/kg/min infusion) and one unfractionated heparin (UFH) group (70-100 IU/kg bolus), 138 patients were analyzed. Argatroban dose-dependently prolonged activated clotting time (ACT) with more patients reaching the minimum target ACT after the initial bolus injection (ARG250: 86.1%, ARG300: 89.5%, and ARG350: 96.8%) compared to 45.5% in UFH (p<0.001). The patient proportion who did not require additional bolus injections to start PCI was significantly higher in argatroban than in UFH (p ≤ 0.002). Consequently, the time to start of PCI was shortened in argatroban groups. Composite incidences of death, myocardial infarction, and urgent revascularization until day 30 were not significantly different between the groups (ARG250: 2.8%, ARG300: 0.0%, ARG350: 3.2% vs. UFH: 3.0%). Major bleeding was observed only in UFH (3.0%), while minor bleeding occurred in ARG350 (3.2%) and UFH (6.1%, n.s.). CONCLUSION: Argatroban dose-dependently increases coagulation parameters and, compared to UFH, demonstrates a superior predictable anticoagulant effect in patients undergoing elective PCI.

[Challenge in diabetes therapy: effects of glitazones beyond blood glucose control]. / Herausforderung Diabetestherapie: Effekte der Glitazone jenseits der Blutzuckerkontrolle.

Not just since the results of ACCORD, ADVANCE and VADT were published, it is clear that lowering blood glucose alone does not reduce the cardiovascular risk of patients with type 2 diabetes. In fact, many studies also indicate that some treatment strategies may even have adverse effects. To treat type 2 diabetes appropriately, the co-morbidities such as diabetic dyslipidaemia, hypertension or nephropathy must also be taken into account. Thiazolidinediones reduce insulin resistance thus allowing to direct the treatment of type 2 diabetes towards its pathophysiologic origin. Due to their mechanism of action, thiazolidinediones not only lower blood glucose but have also beneficial effects on inflammatory and atherogenic parameters, blood pressure and microalbuminuria. Furthermore pioglitazone improves dyslipidaemia and reduces mortality, myocardial infarction and stroke in high risk patients. Effects of rosiglitazone on the cardiovascular risk are yet unclear. Numerous studies document the efficacy and safety of thiazolidinediones and provide a basis for an evidence-based therapeutic approach beyond blood glucose control.

[Treatment of the acute coronary syndrome in Germany: experiences in a German cluster of the GRACE registry]. / Behandlung des akuten Koronarsyndroms in Deutschland: Erfahrungen eines deutschen Clusters des GRACE-Registers.

BACKGROUND: The acute coronary syndrome (ACS) remains a major cause of mortality and morbidity in the western world. The Global Registry of Acute Coronary Events (GRACE) documents inpatients with all types of ACS and a follow-up at three months in Germany and worldwide. METHODS: The data of the German Cluster Detmold were compared with data from the worldwide GRACE registry (31,070 patients). Data from 849 patients with ST-elevation myocardial infarction (STEMI), non-ST elevation myocardial infarction (NSTEMI) and unstable angina (UA) were collected from October 2001 to September 2005 in eight participating hospitals in the GRACE2 Cluster Detmold. RESULTS: Compared with the worldwide GRACE data the patients in the Cluster Detmold had longer pre-hospital admission times (STEMI patients < 1 h: 13.9 % vs. 17.0 %; p < 0.05); more frequent interventions (PCI 60.1 % vs. 48.7%; p < 0.001) and less thrombolysis (17.9 vs. 42.5%; p < 0.001) in STEMI patients; more frequent use of platelet inhibitors (clopidogrel and ticlopidine, 93.4 % vs. 89.4%; p < 0.001) and unfractionated heparin (69.8 % vs. 36.5; p < 0.001), and less frequent use of low molecular weight heparin (31.1 % vs. 51.2%; p < 0.001); more frequent use of RAS blocking agents (80.2 vs. 66.6, p < 0.001) and beta blockers (87.4 vs. 78.8, p < 0.001) and less frequent use of lipid lowering agents (23.5 vs. 72.5%; p < 0.001). CONCLUSIONS: Current management of ACS in Germany closely follows the recommendations of the German society of Cardiology. Differences in practice may account for the observed substantially lower event rates in Germany during hospitalization, but there is still room for improvement in the pre-hospital phase und in the degree to which pharmacotherapy is used for secondary prevention.

[Postoperative antithrombotic treatment in diabetic patients]. / Postoperative antithrombotische Therapie bei diabetischen Patienten.

Diabetes mellitus patients after aorto-coronary bypass operation constitute a patient cohort at largely increased risk for secondary coronary events. Antiplatelet agents and antithrombotic agents are applied for secondary prevention. Up to now, secondary prevention has not been addressed specifically in the cohort of diabetic patients after bypass operation. Hence therapeutic recommendations are derived from the global cohort of CAD patients and based on risk assessment rather than on specific data. Since diabetic patients after myocardial infarction are at particularly high risk, combined therapy with clopidogrel and ASS may be considered even with restricted resources in the health system. Oral anticoagulation with coumadin constitutes an effective alternative to dual anti-platelet therapy. Under specific conditions (ventricular aneurysms, EF < 30%, or certain conditions in coronary anatomy) oral anticoagulants should be considered more liberally than currently.

[Coronary artery disease--relevance of total coronary revascularization on the incidence of malignant arrhythmias]. / Koronare Herzkrankheit--Bedeutung der Ischämiebeseitigung für das Arrhythmieverhalten.

Myocardial ischemia induces redistribution of different ions (H(+), K(+), Na(+), Ca(++)) across the cardiomyocyte membrane, as well as the loss of intracellular ATP content. This results in changes in the electrical properties including shortening of the action potential, appearance of delayed afterpotentials, and a modified refractoriness of the cardiomyocyte. These changes may induce or support malignant cardiac arrhythmias. Supersensitivity of sympathetic denervated myocardium may further support the electrical instability of ischemic myocardium.Virtues of studies indicate that patients with coronary artery disease who develop complex arrhythmias during or after exercise bear a substantially increased risk for sudden cardiac death. Other studies report about arrhythmic stabilization and reduced mortality if patients with reversible myocardial ischemia receive complete revascularization. However, none of these studies is without methodological flaws. Due to the lack of methodologically sound studies in sufficiently large patient cohorts, the question whether complete coronary revascularisation improves the prognosis of patients with coronary artery disease and which strategy (medical, interventional, or surgical) warrants the best outcomes remains open.

Creatinine clearance and adverse hospital outcomes in patients with acute coronary syndromes: findings from the global registry of acute coronary events (GRACE).

OBJECTIVE: To determine whether creatinine clearance at the time of hospital admission is an independent predictor of hospital mortality and adverse outcomes in patients with acute coronary syndromes (ACS). DESIGN: A prospective multicentre observational study, GRACE (global registry of acute coronary events), of patients with the full spectrum of ACS. SETTING: Ninety four hospitals of varying size and capability in 14 countries across four continents. PATIENTS: 11 774 patients hospitalised with ACS, including ST and non-ST segment elevation acute myocardial infarction and unstable angina. MAIN OUTCOME MEASURES: Demographic and clinical characteristics, medication use, and in-hospital outcomes were compared for patients with creatinine clearance rates of > 60 ml/min (normal and minimally impaired renal function), 30-60 ml/min (moderate renal dysfunction), and < 30 ml/min (severe renal dysfunction). RESULTS: Patients with moderate or severe renal dysfunction were older, were more likely to be women, and presented to participating hospitals with more comorbidities than those with normal or minimally impaired renal function. In comparison with patients with normal or minimally impaired renal function, patients with moderate renal dysfunction were twice as likely to die (odds ratio 2.09, 95% confidence interval 1.55 to 2.81) and those with severe renal dysfunction almost four times more likely to die (odds ratio 3.71, 95% confidence interval 2.57 to 5.37) after adjustment for other potentially confounding variables. The risk of major bleeding episodes increased as renal function worsened. CONCLUSION: In patients with ACS, creatinine clearance is an important independent predictor of hospital death and major bleeding. These data reinforce the importance of increased surveillance efforts and use of targeted intervention strategies in patients with acute coronary disease complicated by renal dysfunction.

Urokinase plasminogen activator augments cell proliferation and neointima formation in injured arteries via proteolytic mechanisms.

Urokinase plasminogen activator (uPA) has been implicated in the healing responses of injured arteries, but the importance of its various properties that influence smooth muscle cell (SMC) proliferation and migration in vivo is unclear. We used three recombinant (r-) forms of uPA, which differ markedly in their proteolytic activities and abilities to bind to the uPA receptor (uPAR), to determine, which property most influences the healing responses of balloon catheter injured rat carotid arteries. After injury, uPA and uPAR expression increased markedly throughout the period when medial SMCs were rapidly proliferating and migrating to form the neointima. Perivascular application of uPA neutralizing antibodies immediately after injury attenuated the healing response, significantly reducing neointima size and neointimal SMC numbers. Perivascular application of r-uPAwt (wild type uPA) or r-uPA/GDF (r-uPA with multiple mutations in its growth factor-like domain) doubled the size of the neointima. Four days after injury these two uPAs nearly doubled neointimal and medial SMC numbers in the vessels, and induced greater reductions in lumen size than injury alone. Proteolytically inactive r-uPA/H/Q (containing glutamine rather than histidine-204 in its catalytic site) did not affect neointima or lumen size. Also, in contrast to the actions of proteolytically active uPAs, tissue plasminogen activator (tPA) did not affect the rate of neointima development. We conclude that uPA is an important factor regulating the healing responses of balloon catheter injured arteries, and its proteolytic property, which cannot be mimicked by tPA, greatly influences SMC proliferation and early neointima formation.

Ecarin clotting time but not aPTT correlates with PEG-hirudin plasma activity.

BACKGROUND: Novel antithrombotic agents such as hirudin have shown promise in the therapy of acute coronary syndromes. PEG-hirudin (polyethyleneglycol conjugated hirudin) has been developed to provide a longer plasma half-life and more stable antithrombotic plasma levels. Privious trials indicated a narrow therapeutic window for hirudin and a number of aPTT (activated partial thromboplastin time)-monitored trials investigating hirudin in acute coronary syndromes had to be stopped because of intracranial bleeding complications. OBJECTIVES: The present study evaluates the ecarin clotting time (ECT), a parameter based on the conversion of prothrombin by the snake venom enzyme ecarin, for the monitoring of PEG-hirudin therapy. METHODS: Plasma from either healthy volunteers (n=20) or from patients (n=10) suffering from unstable angina pectoris (UAP) was spiked with increasing PEG-hirudin concentrations. In a prospective randomized clinical trial patients with UAP were treated with intravenous PEG-hirudin or heparin over 72 hours. Patients were randomized to the following treatment groups: (1) heparin control group, n=15; (2) PEG-hirudin low dose (0.1 mg/kg bolus, 0.01 mg/kg/h infusion), n=19; (3) intermediate dose (0.15 mg/kg and 0.015 mg/kg/h), n=17; 4) high-dose (0.2 mg/kg and 0.02 mg/kg/h), n=16. Spiked plasma samples and plasma from UAP patients treated with i.v. PEG-hirudin were analyzed for aPTT, ECT, and PEG-hirudin levels. RESULTS: A linear correlation up to the highest therapeutic concentrations could be observed between PEG-hirudin plasma concentrations and the ECT. This was true for both plasma samples spiked with PEG-hirudin in vitro as well as for samples taken from patients treated with i.v. PEG-hirudin (correlation coefficient 0.9, respect.) In contrast the aPTT did not show a reliable linear correlation to PEG-hirudin concentrations. CONCLUSION: Monitoring of PEG-hirudin therapy by ECT may help to avoid inadequate anticoagulation or overdosing. Thus, the safety and efficacy profile of PEG-hirudin therapy is likely to be enhanced by ECT monitoring.

Plasminogen activator inhibitor type-1 in cardiovascular disease. Status report 2001.

Plasminogen activator inhibitor type-1 (PAI-1) is known to contribute to thrombus formation and to the development and the clinical course of acute and chronic cardiovascular disease, as well as of other arterial and venous thromboembolic diseases. Recently, an important role of elevated pretreatment levels of PAI-1 for failure of thrombolytic therapy of acute myocardial infarction has been discussed. PAI-1 plasma levels depend on the one hand on gene regulation but are related on the other hand to known risk factors of atherosclerosis like insulin resistance, diabetes or hypertriglyceridemia, respectively. Furthermore, an activated renin-angiotensin-aldosterone system (RAAS) significantly contributes to the upregulation of PAI-1 concentration via a receptor-mediated mechanism. In accordance to the known mechanisms of regulation of PAI-1 plasma levels, the use of specific agents like antidiabetic drugs, fibrates, statins, ACE inhibitors and angiotensin II type-1 receptor-blockers may contribute to the downregulation of circulating PAI-1 and, therefore, increase the fibrinolytic capacity and consecutively counteract the thrombotic tendency. To further improve the efficacy of thrombolytic therapy, a PAI-1 resistant variant of t-PA, TNK-t-PA, has been developed and is now available for acute myocardial infarction.

Amelioration of angiotensin II-induced cardiac injury by a 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitor.

BACKGROUND: 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have effects that extend beyond cholesterol reduction. We used an angiotensin (Ang) II-dependent model to test the hypothesis that cerivastatin ameliorates cardiac injury. METHODS AND RESULTS: We treated rats transgenic for human renin and angiotensinogen (dTGR) chronically from weeks 4 to 7 with cerivastatin (0.5 mg/kg by gavage). We used immunohistochemistry, electrophoretic mobility shift assays, and reverse transcription-polymerase chain reaction techniques. Compared with control dTGR, dTGR treated with cerivastatin had reduced mortality, blood pressure, cardiac hypertrophy, macrophage infiltration, and collagen I, laminin, and fibronectin deposition. Basic fibroblast growth factor mRNA and protein expression were markedly reduced, as was interleukin-6 expression. The transcription factors NF-kappaB and AP-1 were substantially less activated, although plasma cholesterol was not decreased. CONCLUSIONS: These results suggest that statins ameliorate Ang II-induced hypertension, cardiac hypertrophy, fibrosis, and remodeling independently of cholesterol reduction. Although the clinical significance remains uncertain, the results suggest that statins interfere with Ang II-induced signaling and transcription factor activation, thereby ameliorating end-organ damage.

Anticoagulation: the present and future.

Thrombin is a central bioregulator of coagulation and is therefore a key target in the therapeutic prevention and treatment of thromboembolic disorders, including deep vein thrombosis and pulmonary embolism. The current mainstays of anticoagulation treatment are heparins, which are indirect thrombin inhibitors, and coumarins, such as warfarin, which modulate the synthesis of vitamin K-dependent proteins. Although efficacious and widely used, heparins and coumarins have limitations because their pharmacokinetics and anticoagulant effects are unpredictable, with the risk of bleeding and other complications resulting in the need for close monitoring with their use. Low-molecular-weight heparins (LMWHs) provide a more predictable anticoagulant response, but their use is limited by the need for subcutaneous administration. In addition, discontinuation of heparin treatment can result in a thrombotic rebound due to the inability of these compounds to inhibit clot-bound thrombin. Direct thrombin inhibitors (DTI) are able to target both free and clot-bound thrombin. The first to be used was hirudin, but DTIs with lower molecular weights, such as DuP 714, PPACK, and efegatran, have subsequently been developed, and these agents are better able to inhibit clot-bound thrombin and the thrombotic processes that take place at sites of arterial damage. Such compounds inhibit thrombin by covalently binding to it, but this can result in toxicity and nonspecific binding. The development of reversible noncovalent DTIs, such as inogatran and melagatran, has resulted in safer, more specific and predictable anticoagulant treatment. Oral DTIs, such as ximelagatran, are set to provide a further breakthrough in the prophylaxis and treatment of thrombosis.

[Significance of the uPA/uPAR system for development of arteriosclerosis and restenosis]. / Die Bedeutung des uPA/uPAR-Systems für die Entwicklung von Arteriosklerose und Restenose.

Arteriosclerosis and the development of restenosis still remain a significant clinical problem. Migration of vascular smooth muscle cells from the media to the intima and cell proliferation are the hallmarks of the underlying pathomechanisms. Cell migration requires chemotaxis, phenotypic changes of cells, cell adhesive and de-adhesive events and the coordinated remodeling of the extracellular matrix. One of the phenotypic changes induced in migrating cells is the increased expression of urokinase plasminogen activator (uPA) and of its specific receptor uPAR. They are polarized to the leading edge of migrating cells. Both uPA and uPAR are key mediators of extracellular proteolysis. They participate in extracellular matrix remodeling, activate cells and enable them to migrate and invade into different tissue layers. UPA/uPAR are multifunctional proteins influencing a great variety of signal transduction pathways ultimately culminating in the regulation of cell migration and proliferation. In addition to time- and space-confined proteolysis this powerful system can mediate chemotaxis, cell adhesion and gene expression, partly by interacting in concert with integrins, G proteins, or with yet unidentified coreceptors or adapter molecules. Interaction with the uPA/uPAR system or components of its specific signal transduction pathways may serve as a guide for the development of effective therapeutic strategies to prevent arteriosclerosis and restenosis after percutaneous arterial angioplastic interventions.

[Direct thrombin antagonists]. / Direkte Thrombinantagonisten.

BACKGROUND: Inhibiting thrombin as a key enzyme of the coagulation cascade is therapeutically useful in thromboembolic diseases. In coronary thrombosis, direct thrombin inhibitors promise to be useful for an efficacious therapy. Hirudin and recombinant or synthetic mimetics like hirulog, argatroban and melagatran have proven their efficacy in clinical studies. CONCLUSION: Therapy with direct thrombin inhibitors such as hirudin and analogous substances reduces coronary events. Moreover, the agents are useful for therapy of thromboembolic diseases, especially in the case of heparin induced thrombocytopenia type II.

[Recommendations of the International Cardiology Forum (ICF) 1998 on revision of the current guidelines for the diagnosis and treatment of unstable angina pectoris and nontransmural myocardial infarct]. / Empfehlungen des International Cardiology Forum (ICF) 1998 zur Revision der bestehenden Richtlinien zur Diagnostik und Therapie der instabilen Angina pectoris und des nicht-transmuralen Myokardinfarktes.

Rapid progress in the understanding and treatment of unstable angina and acute coronary syndrome are prompting occasional revisions to current treatment guidelines. The recommendations contained in this paper are based on the consensus reached during discussions at the 'International Cardiology Forum' in September 1998. Consensus was reached on significant major points, although some aspects remain controversial. A substantial body of data accumulated in a host of studies justify changes to current treatment habits.

Urokinase stimulates human vascular smooth muscle cell migration via a phosphatidylinositol 3-kinase-Tyk2 interaction.

Janus kinases Jak1 and Tyk2 play an important role in urokinase-type plasminogen activator (uPA)-dependent signaling. We have recently demonstrated that both kinases are associated with the uPA receptor (uPAR) and mediate uPA-induced activation of signal transducers and activators of transcription (Stat1, Stat2, and Stat4) in human vascular smooth muscle cells (VSMC). Janus kinases are not only required for Stat activation but may also interfere with other intracellular signaling pathways. Here we report that in VSMC, Tyk2 interacts with a downstream signaling cascade involving phosphatidylinositol 3-kinase (PI3-K). We demonstrate that uPA induces PI3-K activation, which is abolished in VSMC expressing the dominant negative form of Tyk2. The regulatory subunit p85 of PI3-K co-immunoprecipitates with Tyk2 but not with Jak1, Jak2, or Jak3, and uPA stimulation increases the PI3-K activity in Tyk2 immunoprecipitates. Tyk2 directly binds to either of the two Src homology 2(SH2)p85 domains in a uPA-dependent fashion. We provide evidence that the Tyk2-mediated PI3-K activation in response to uPA is required for VSMC migration. Thus, two unrelated structurally distinct specific inhibitors of PI3-K, wortmannin and LY294002, prevent VSMC migration induced by uPA. No migratory effect of uPA was observed in VSMC expressing the dominant negative form of Tyk2. Our results underscore the versatile function of Tyk2 in uPA-related intracellular signaling and indicate that PI3-K plays a selective role in the regulation of VSMC migration.

Angiotensin II (AT(1)) receptor blockade reduces vascular tissue factor in angiotensin II-induced cardiac vasculopathy.

Tissue factor (TF), a main initiator of clotting, is up-regulated in vasculopathy. We tested the hypothesis that chronic in vivo angiotensin (ANG) II receptor AT(1) receptor blockade inhibits TF expression in a model of ANG II-induced cardiac vasculopathy. Furthermore, we explored the mechanisms by examining transcription factor activation and analyzing the TF promoter. Untreated transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR) feature hypertension and severe left ventricular hypertrophy with focal areas of necrosis, and die at age 7 weeks. Plasma and cardiac ANG II was three- to fivefold increased compared to Sprague-Dawley rats. Chronic treatment with valsartan normalized blood pressure and coronary resistance completely, and ameliorated cardiac hypertrophy (P < 0.001). Valsartan prevented monocyte/macrophage infiltration, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) activation, and c-fos expression in dTGR hearts. NF-kappaB subunit p65 and TF expression was increased in the endothelium and media of cardiac vessels and markedly reduced by valsartan treatment. To analyze the mechanism of TF transcription, we then transfected human coronary artery smooth muscle cells and Chinese hamster ovary cells overexpressing the AT(1) receptor with plasmids containing the human TF promoter and the luciferase reporter gene. ANG II induced the full-length TF promoter in both transfected cell lines. TF transcription was abolished by AT(1) receptor blockade. Deletion of both AP-1 and NF-kappaB sites reduced ANG II-induced TF gene transcription completely, whereas the deletion of AP-1 sites reduced transcription. Thus, the present study clearly shows an aberrant TF expression in the endothelium and media in rats with ANG II-induced vasculopathy. The beneficial effects of AT(1) receptor blockade in this model are mediated via the inhibition of NF-kappaB and AP-1 activation, thereby preventing TF expression, cardiac vasculopathy, and microinfarctions.

AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor.

BACKGROUND: We recently described autoantibodies (angiotensin-1 receptor autoantibodies, AT(1)-AA) directed at the AT(1) receptor in the serum of preeclamptic patients, whose placentas are commonly infarcted and express tissue factor (TF). Mechanisms of how AT(1)-AA might contribute to preeclampsia are unknown. We tested the hypothesis that AT(1)-AA cause vascular smooth muscle cells (VSMC) to express TF. METHODS AND RESULTS: IgG from preeclamptic patients containing AT(1)-AA was purified with anti-human IgG columns. AT(1)-AA were separated from the IgG by ammonium sulfate precipitation. We transfected Chinese hamster ovary cells overexpressing the AT(1) receptor with TF promoter constructs coupled to a luciferase reporter gene. VSMC were obtained from human coronary arteries. Extracellular signal-related kinase activation was detected by an in-gel kinase assay. AP-1 activation was determined by electromobility shift assay. TF was measured by ELISA and detected by immunohistochemistry. Placentas from preeclamptic women stained strongly for TF, whereas control placentas showed far less staining. We proved AT(1)-AA specificity by coimmunoprecipitating the AT(1) receptor with AT(1)-AA but not with nonspecific IgG. Angiotensin (Ang) II and AT(1)-AA both activated extracellular signal-related kinase, AP-1, and the TF promoter transfected VSMC and Chinese hamster ovary cells, but only when the AP-1 binding site was present. We then demonstrated TF expression in VSMC exposed to either Ang II or AT(1)-AA. All these effects were blocked by losartan. Nonspecific IgG or IgG from nonpreeclamptic pregnant women had a negligible effect. CONCLUSIONS: We conclude that AT(1)-AA and Ang II both stimulate the AT(1) receptor and initiate a signaling cascade resulting in TF expression. These results show an action of AT(1)-AA on human cells that could contribute to the pathogenesis of preeclampsia.

Urokinase-induced mitogenesis is mediated by casein kinase 2 and nucleolin.

BACKGROUND: Urokinase (uPA) and the urokinase receptor (uPAR) form a multifunctional system capable of concurrently regulating pericellular proteolysis, cell-surface adhesion, and mitogenesis. The role of uPA and uPAR in directed proteolysis is well established and its function in cellular adhesiveness has recently been clarified by numerous studies. The molecular mechanisms underlying the mitogenic effects of uPA and uPAR are still unclear, however. RESULTS: We identified mechanisms that might participate in uPA-related mitogenesis in human vascular smooth muscle cells and demonstrated that uPA induces activation of a unique signaling complex. This complex contains uPAR and two additional proteins, nucleolin and casein kinase 2, which are implicated in cell proliferation. Both proteins were isolated by affinity chromatography on uPA-conjugated cyanogen-bromide-activated Sepharose 4B and were identified using nano-electrospray mass spectrometry and immunoblotting. We used laser scanning and immunoelectron microscopy studies to further demonstrate that nucleolin and casein kinase 2 are located on the cell surface where they colocalize with the uPAR. Moreover, the proteins were co-internalized into the cell as an entire complex. Immunoprecipitation experiments in combination with an in vitro kinase assay demonstrated a specific association of uPAR with nucleolin and casein kinase 2 and revealed a uPA-induced activation of casein kinase 2, which presumably led to phosphorylation of nucleolin. Blockade of nucleolin and casein kinase 2 with specific modulators led to the inhibition of uPA-induced cell proliferation. CONCLUSIONS: We conclude that in human vascular smooth muscle cells, uPA induces the formation and activation of a newly identified signaling complex comprising uPAR, nucleolin, and casein kinase 2, that is responsible for the uPA-related mitogenic response. The complex is not a unique feature of vascular smooth muscle cells, as it was also found in other uPAR-expressing cell types.